Production of protein extracts from Swedish red, green, and brown seaweeds, Porphyra umbilicalis Kützing, Ulva lactuca Linnaeus, and Saccharina latissima (Linnaeus) J. V. Lamouroux using three different methods

peer-reviewedThe demand for vegetable proteins increases globally and seaweeds are considered novel and promising protein sources. However, the tough polysaccharide-rich cell walls and the abundance of polyphenols reduce the extractability and digestibility of seaweed proteins. Therefore, food grade, scalable, and environmentally friendly protein extraction techniques are required. To date, little work has been carried out on developing such methods taking into consideration the structural differences between seaweed species. In this work, three different protein extraction methods were applied to three Swedish seaweeds (Porphyra umbilicalis, Ulva lactuca, and Saccharina latissima). These methods included (I) a traditional method using sonication in water and subsequent ammonium sulfate-induced protein precipitation, (II) the pH-shift protein extraction method using alkaline protein solubilization followed by isoelectric precipitation, and (III) the accelerated solvent extraction (ASE®) method where proteins are extracted after pre-removal of lipids and phlorotannins. The highest protein yields were achieved using the pH-shift method applied to P. umbilicalis (22.6 ± 7.3%) and S. latissima (25.1 ± 0.9%). The traditional method resulted in the greatest protein yield when applied to U. lactuca (19.6 ± 0.8%). However, the protein concentration in the produced extracts was highest for all three species using the pH-shift method (71.0 ± 3.7%, 51.2 ± 2.1%, and 40.7 ± 0.5% for P. umbilicalis, U. lactuca, and S. latissima, respectively). In addition, the pH-shift method was found to concentrate the fatty acids in U. lactuca and S. latissima by 2.2 and 1.6 times, respectively. The pH-shift method can therefore be considered a promising strategy for producing seaweed protein ingredients for use in food and feed

Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models

In this work, we investigated lipid oxidation of cod liver oil during gastrointestinal (GI) digestion using two types of in vitro digestion models. In the first type of model, we used human GI juices, while we used digestive enzymes and bile from porcine origin in the second type of model. Human and porcine models were matched with respect to factors important for lipolysis, using a standardized digestion protocol. The digests were analysed for reactive oxidation products: malondialdehyde (MDA), 4-hydroxy-trans-2-nonenal (HNE), and 4-hydroxy-trans-2-hexenal (HHE) by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS), and for free fatty acids (FFA) obtained during the digestion by gas chromatography-mass spectrometry (GC-MS). The formation of the oxidation products MDA, HHE, and HNE was low during the gastric digestion, however, it increased during the duodenal digestion. The formation of the oxidation products reached higher levels when digestive juices of human origin were used (60 μM of MDA, 0.96 μM of HHE, and 1.6 μM of HNE) compared to when using enzymes and bile of porcine origin (9.8, and 0.36 μM of MDA; 0.16, and 0.026 μM of HHE; 0.23, and 0.005 μM of HNE, respectively, in porcine models I and II). In all models, FFA release was only detected during the intestinal step, and reached up to 31% of total fatty acids (FA). The findings in this work may be of importance when designing oxidation oriented lipid digestion studies

Circulating Linoleic Acid is Associated with Improved Glucose Tolerance in Women after Gestational Diabetes

Abstract: Women with previously diagnosed gestational diabetes mellitus (GDM) are at increasedrisk of type-2-diabetes mellitus (T2D).We aimed to establish links between glucose tolerance (GT)and serum fatty acid (FA) profile in the transition from GDM to T2D. Six years after GDM, 221 womenwere grouped as having normal GT (NGT), impaired GT (IGT), or T2D based on oral GT testresults. Fasting serum FAs were profiled, anthropometric measures taken, and dietary intakedetermined. Linoleic acid (LA) was significantly higher in NGT women (p < 0.001) compared withIGT and T2D, and emerged as a strong predictor of low glucose and insulin levels, independentlyof BMI. Self-reported vegetable oil consumption correlated with LA serum levels and glucoselevels. Delta-6-, delta-9-, and stearoyl-CoA-desaturase activities were associated with decreased GT,and delta-5-desaturase activities with increased GT. In a subgroup of women at high risk of diabetes,low LA and high palmitic acid levels were seen in those that developed T2D, with no differences inother FAs or metabolic measurements. Results suggest that proportions of LA and palmitic acid areof particular interest in the transition from GDM to T2D. Interconversions between individual FAsregulated by desaturases appear to be relevant to glucose metabolis

Effect of stabilization method and freeze/thaw-aided precipitation on structural and functional properties of proteins recovered from brown seaweed (Saccharina latissima)

Structural, functional and nutritional properties of protein recovered from brown seaweed,\ua0S. latissima\ua0with alkaline solubilization/isoelectric precipitation as a function of different post-harvest stabilization methods were studied. The latter included freezing at −20 \ub0C/-80 \ub0C, oven-drying, sun-drying, freeze-drying and ensilaging. Also, the efficacy of freeze/thaw-aided precipitation (F/T) in improving protein recovery of the process was evaluated. The freeze-dried, oven-dried, and −20 \ub0C frozen seaweeds resulted in significantly higher protein yield than the −80\ub0C-frozen, sun-dried and ensiled biomasses. F/T increased protein precipitation and doubled total protein yield. Sun-drying and −20\ub0C-freezing caused extensive protein degradation as revealed by SDS-PAGE and HP-SEC, while oven-drying altered the seaweed protein structure with less α-helices. Functional properties of the seaweed proteins were remarkably affected by stabilization condition and F/T, but nutritional value of the proteins was only dependent on stabilization method. Thus, to efficiently recover seaweed proteins, its post-harvest stabilization condition must be carefully chosen based on the final application of the proteins

Fatty acid contaminations originating from commercially available solid phase-extraction columns

It has been shown previously that commercially available solid phase extraction (SPE) columns release contaminants such as palmitic acid and stearic acid during separation of lipid classes. The presence of contaminating fatty acids in the fatty acid fraction is particularly troublesome. We here confirm that the overwhelming majority of the contaminants originate from the barrels, and have identified two contaminants as palmitic acid and stearic acid or their equivalents. We urge readers to take fatty acid contaminants into careful consideration when planning their experiments, and if necessary use sorbent packed in glass barrels instead of plastic such as polypropylene

myo-Inositol phosphate isomers generated by the action of a phytate-degrading enzyme from Klebsiella terrigena on phytate

For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P-5, D-Ins(1,2,5,6)P-4, D-Ins(1,2,6)P-3, D-Ins(1,2)P-2 and alternatively via D-Ins(1,2,4,5,6)P-5, Ins(2,4,5,6)P-4, D-Ins(2,4,5)P-3, D-Ins(2,4)P-2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P-5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P-5, D-Ins(1,2,3,6)P-4, Ins(1,2,3)P-3, D-Ins(2,3)P-2 and alternatively via D-Ins(1,2,3,5,6)P-5, D-Ins(2,3,5,6)P-4, D-Ins(2,3,5)P-3, D-Ins(2,3)P-2 to finally Ins(2)P. D-Ins(2,3,5,6)P-4, D-Ins(2,3,5)P-3, and D-Ins(2,4)P-2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P-5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.Key words: myo-inositol phosphate isomers, phytate-degrading enzyme, phytate, phytase, Klebsiella terrigena

Assessing phytase activity–methods, definitions and pitfalls.

Phytases are nutritionally important for increased bioavailability of dietary minerals and phosphate for monogastric animals including humans. Release of minerals and phosphate is accomplished by the enzymatic stepwise degradation of phytate (inositol hexaphosphate, IP6). Activity determinations of phytase is often based on analysis of total released phosphate (Pi), but phytase activity in its purest form represents released product per time from IP6 only. Microbial and plant preparations often also contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from non-phytase phosphatases degrading non-phytate molecules. Moreover, even purified enzyme extracts assessed via Pi release may result in errors, since commercial IP6 commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP6 hydrolysis are also substrates for the phytase. These facts motivate a quantitative comparative study. We compared enzyme activity determination in phytase assay samples at four different time points, based on analyzing the substrate IP6 versus the product Pi using different selected methods. The calculated activities varied substantially. For example, at 15 min into enzymatic assay, variations from 152 mU/ml (by IP6 analysis on HPIC) to 275-586 mU/ml (by Pi analysis using several methods) was detected. Our work emphasizes the importance of defining the type of activity assessed, showing that phytase activity based on released Pi may yield false positive results and/or overestimations. We propose to differentiate between phytase activity, being the activity by which IP6 is degraded, and total inositol phosphatase activity, corresponding to total released phosphate during the enzymatic reaction

Quantification of total fatty acids in microalgae: comparison of extraction and transesterification methods

Determination of microalgaes\u27 fatty acid content is often done with chloroform and methanol according to the Bligh and Dyer extraction, though faster methods exist. A number of comparisons between the Bligh and Dyer and faster methods have resulted in contradicting data, possibly due to differences in algae used and the different versions of the Bligh and Dyer method applied. Here, various forms of direct-transesterification (D-TE) and two-step transesterification (2-TE), including three versions developed in our lab, are compared with the original Bligh and Dyer (Can J Biochem Physiol 37: 911-917, 1959) extraction and two modifications thereof (Lee et al. J AOAC Int 79:487-492, 1996, and our own acidified version) on microalgae with different cell walls: Isochrysis galbana, Nannochloropsis oculata, and Phaeodactylum tricornutum. In total, fatty acid extracts from 11 methods were separated and quantified by gas chromatography with mass spectrometry. Results show that, for N. oculata and P. tricornutum, methods based on chloroform-methanol underestimated the fatty acid content compared with the 2-TE and D-TE methods, which gave similar results. Moreover, D-TE methods are faster than chloroform-methanol methods and use chemicals that are less toxic. Of the D-TE methods, the ones using hydrochloric acid and sulfuric acid recovered the most fatty acids, while boron trifluoride recovered slightly less. The main qualitative difference between the fatty acids recovered was that the chloroform-methanol methods recovered less saturated fatty acids in P. tricornutu

Assessing phytase activity–methods, definitions and pitfalls

Phytases are nutritionally important for increased bioavailability of dietary minerals and phosphate for monogastric animals including humans. Release of minerals and phosphate is accomplished by the enzymatic stepwise degradation of phytate (inositol hexaphosphate, IP₆). Activity determinations of phytase is often based on analysis of total released phosphate (P_i), but phytase activity in its purest form represents released product per time from IP₆ only. Microbial and plant preparations often also contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from non-phytase phosphatases degrading non-phytate molecules. Moreover, even purified enzyme extracts assessed via P_i release may result in errors, since commercial IP₆ commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP₆ hydrolysis are also substrates for the phytase. These facts motivate a quantitative comparative study. We compared enzyme activity determination in phytase assay samples at four different time points, based on analyzing the substrate IP₆ versus the product P_i using different selected methods. The calculated activities varied substantially. For example, at 15 min into enzymatic assay, variations from 152 mU/ml (by IP₆ analysis on HPIC) to 275-586 mU/ml (by P_i analysis using several methods) was detected. Our work emphasizes the importance of defining the type of activity assessed, showing that phytase activity based on released P_i may yield false positive results and/or overestimations. We propose to differentiate between <em>phytase activity</em>, being the activity by which IP₆ is degraded, and <em>total inositol phosphatase activity</em>, corresponding to total released phosphate during the enzymatic reaction